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MD simulation of sensory rhodopsin II was executed for three intermediates (ground-state, K-state, M-state) appearing in its photocycle. We observed a large displacement of the cytoplasmic side of helixF only in M-state among the three intermediates. This displacement was transmitted to TM2, and the cytoplasmic side of TM2 rotated clockwise. These transient movements are in agreement with the results of an EPR experiment. That is, the early stage of signal transduction in a sRII-HtrII complex was successfully reproduced by the in silico MD simulation. By analyzing the structure of the sRII-HtrII complex, the following findings about the photocycle of sRII were obtained: (1) The hydrogen bonds between helixF and other helices determine the direction of the movement of helixF; (2) three amino acids (Arg162, Thr189, Tyr199) are essential for sRII-HtrII binding and contribute to the motion transfer from sRII to HtrII; (3) after the isomerization of retinal, a major conformational change of retinal was caused by proton transfer from Schiff base to Asp75, which, in turn, triggers the steric collision of retinal with Trp171. This is the main reason for the movement of the cytoplasmic side of helixF. [ABSTRACT FROM AUTHOR]

Abstract: The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model. [Copyright &y& Elsevier]

Abstract: The alternative NADH:ubiquinone oxidoreductase (NDH-2) from Escherichia coli is a membrane protein playing a prominent role in respiration by linking the reduction of NADH to the quinone pool. Remote sequence similarity reveals an evolutionary relation between alternative NADH:quinone oxidoreductases and the SCOP-family “FAD/NAD-linked reductases”. We have created a structural model for NDH-2 from E. coli through comparative modelling onto a template from this family. Combined analysis of our model and sequence conservation allowed us to include the cofactor FAD and the substrate NADH in atomic detail. Furthermore, we propose the most plausible orientation of NDH-2 relative to the membrane and specify a region of the protein potentially involved in ubiquinone binding. [Copyright &y& Elsevier]

Uncoupling proteins (UCP) are known to transport anions, such as Cl-, in addition to H+ transport. Although H+ transport by UCP is clearly involved in thermogenesis, the mechanism of its anion transport is not clearly understood. In this study, we examined the anion channel characteristics of the six individual helical transmembrane (TM) domains of the human UCP2. The second TM domain peptide (TM2) forms multi-state channels by assemblies of conductive oligomers. Furthermore, the TM2 exhibited voltage-dependent anion channels with properties comparable to those of UCP1 chloride channel. However, the other five TM peptides did not form UCP1-like channels. Moreover, an analog of TM2 in which two Arg residues were substituted by Ala residues did not form stable channels, implying the significance of Arg residues for anion transport. These results suggest that the anion channel structure of UCP2 protein is oligomeric and the second TM domain is essential for the voltage-dependence of this anion channel. [Copyright &y& Elsevier]

Cell infection by alphaviruses is accompanied by membrane permeability changes. New predictive approaches, including the computation of interfacial affinity and corresponding hydrophobic moments, suggest a segmented amphipathic-at-interface domain in the stem region of Semliki Forest virus fusion protein E1. Expression of E1 sequences in Escherichia coli cells confirmed that the membrane proximal plus transmembrane (TM) domain unit permeabilizes cells as efficiently as the 6K viroporin. Both our predictive and experimental data support the involvement of the E1 stem-TM region in membrane insertion and permeabilization. We propose to combine Wimley–White hydrophobicity analysis with expression-coupled permeability assays in order to identify viral products implied in breaching cell membrane barriers during infection. [Copyright &y& Elsevier]

The Escherichia coli MscS mechanosensitive channel protein has a distinct domain structure that terminates in a conserved seven-strand β barrel. This distinctive feature suggested it could be a critical determinant of channel stability and activity. Measurements on a protein deleted for the base of the vestibule and the β barrel (residues 266–286) suggested that the modified channel had reduced activity. However, induction of the mutant protein resulted in membrane protein accumulation equivalent to wild type and a physiologically functional channel. In patch clamp analysis the activity profile was similar to wild type but reduced numbers of channel were seen per patch, suggesting reduced assembly or stability of the mutant protein. The mutant channel exhibited a subtle change in character – channels did not re-open after full desensitization. Thus the immediate carboxy-terminus (residues 266–286) is not essential for MscS gating but improves stability and activity and is required for recovery of channel activity after desensitization. [Copyright &y& Elsevier]

We propose a new method for classifying and identifying transmembrane (TM) protein functions in proteome-scale by applying a single-linkage clustering method based on TM topology similarity, which is calculated simply from comparing the lengths of loop regions. In this study, we focused on 87 prokaryotic TM proteomes consisting of 31 proteobacteria, 22 gram-positive bacteria, 19 other bacteria, and 15 archaea. Prior to performing the clustering, we first categorized individual TM protein sequences as 'known,' 'putative' (similar to 'known' sequences), or 'unknown' by using the homology search and the sequence similarity comparison against SWISS-PROT to assess the current status of the functional annotation of the TM proteomes based on sequence similarity only. More than three-quarters, that is, 75.7% of the TM protein sequences are functionally 'unknown,' with only 3.8% and 20.5% of them being classified as 'known' and 'putative,' respectively. Using our clustering approach based on TM topology similarity, we succeeded in increasing the rate of TM protein sequences functionally classified and identified from 24.3% to 60.9%. Obtained clusters correspond well to functional superfamilies or families, and the functional classification and identification are successfully achieved by this approach. For example, in an obtained cluster of TM proteins with six TM segments, 109 sequences out of 119 sequences annotated as 'ATP-binding cassette transporter' are properly included and 122 'unknown' sequences are also contained. [ABSTRACT FROM AUTHOR]

Methods that predict the topology of helical membrane proteins are standard tools when analyzing any proteome. Therefore, it is important to improve the performance of such methods. Here we introduce a novel method, PRODIV-TMHMM, which is a profile-based hidden Markov model (HMM) that also incorporates the best features of earlier HMM methods. In our tests, PRODIV-TMHMM outperforms earlier methods both when evaluated on 'low-resolution' topology data and on high-resolution 3D structures. The results presented here indicate that the topology could be correctly predicted for approximately two-thirds of all membrane proteins using PRODIV-TMHMM. The importance of evolutionary information for topology prediction is emphasized by the fact that compared with using single sequences, the performance of PRODIV-TMHMM (as well as two other methods) is increased by approximately 10 percentage units by the use of homologous sequences. On a more general level, we also show that HMM-based (or similar) methods perform superiorly to methods that focus mainly on identification of the membrane regions. [ABSTRACT FROM AUTHOR]

Recent three-dimensional structures of helical membrane proteins present new challenges for the prediction of structure from amino acid sequence. Membrane proteins reside stably in a thermodynamic free energy minimum after release into the membrane’s bilayer fabric from the translocon complex. This means that structure prediction is primarily a problem of physical chemistry. But the folding processes within the translocon must also be considered. A distilled overview of the physical principles of membrane protein stability is presented, and extended to encompass translocon-assisted folding. [Copyright &y& Elsevier]

Lysine-288 in the glucagon-like peptide-1 receptor was predicted to be ideally positioned to play a role in hormone binding. Subsequent mutation of Lys-288 to Ala or Leu greatly reduced hormone affinity, while substitution with Arg had minimal effect. Compared to wild type, the Lys288-Ala receptor had a reduced affinity for three peptide ligands with complete N-terminal sequences but not for their N-truncated analogues. Hence, the role of this positively charged residue, which is conserved at the equivalent position in all other Family B receptors, was determined to be important for receptor interaction with the N-terminal eight residues of peptide agonists. [Copyright &y& Elsevier]

The M2 protein form influenza A virus forms a tetrameric ion channel, which enables proton passage across biological membranes when the N-terminal side is acidified. Among the amino acid residues in the transmembrane domain of the M2 protein, His37 and Trp41 are essential for the pH-regulated proton conductance. Current knowledge about the structures and interactions of His37 and Trp41 suggests a model for the M2 ion channel, in which the channel is closed by a network of His37 hydrogen bonds at neutral pH and is opened by a His37-Trp41 cation–π interaction at acidic pH. [Copyright &y& Elsevier]

The M2 ion channel is an essential component of the influenza A virus. This low-pH gated channel has a high selectivity for protons. Evidence from various experimental data has indicated that the essential structure responsible for the channel is a parallel homo-tetrameric α-helix bundle having a left-handed twist with each helix tilted with respect to the membrane normal. A backbone structure has been determined by solid state nuclear magnetic resonance (NMR). Though detailed structures for the side chains are not available yet, evidence has indicated that His37 and Trp41 in the α-helix are implicated in the local molecular structure responsible for the selectivity and channel gate. More definitive conformations for the two residues were recently suggested based on the known backbone structure and recently obtained NMR data. While two competitive proton-conductance mechanisms have been proposed, the actual proton-conductance mechanism remains an unsolved problem. Computer simulations of an excess proton in the channel and computational studies of the His37/Trp41 conformations have provided insights into these structural and mechanism issues. [Copyright &y& Elsevier]

Human Bfl-1 is an anti-apoptotic Bcl-2 family member. Here, we found that Bfl-1 was converted into a potent death-promoting protein by green fluorescent protein (GFP) fusion with its N-terminus. The transient expression of GFP-Bfl-1 induced cytochrome c release and triggered apoptosis in 293T cells, which depended on the mitochondrial localization of GFP-Bfl-1. Apoptosis induced by GFP-Bfl-1 was significantly blocked by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone, but was not blocked by either Bcl-xL or Bfl-1. Our findings provide a useful model for understanding the structural basis of Bcl-2 family proteins that act in an opposite way despite sharing structural similarity between anti-apoptotic and pro-apoptotic proteins. [Copyright &y& Elsevier]

To gain insight into presenilin-1 (PS1) structural aspects, we explored the structure–function relationship of its N- and C-terminal (NTF and CTF, respectively) complexes. We demonstrated that both NTF and CTF act as independent but inter-changing binding units capable of binding each other (NTF/CTF) or their homologues (NTF/NTF; CTF/CTF). The Alzheimer’s disease-associated PS1 mutations Y115H and M146L do not affect their ability to hetero- and/or homodimerize, thus conserving their basic integrity and function(s). These results suggest that PS1 associates intra-molecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or γ-secretase-associated activity. [Copyright &y& Elsevier]

EMR2 is a human myeloid-restricted member of the EGF-TM7 receptor family that contains a highly conserved G protein-coupled receptor proteolysis site (GPS) in the membrane-proximal region. Here the post-translational proteolytic cleavage of EMR2 at GPS was investigated. We show the cleavage occurs at Leu517-Ser518 and is independent of the transmembrane domains. The non-covalent association of the resulting extracellular α-subunit and transmembrane β-subunit requires a minimum of eight amino acids in the β-subunit. The GPS motif is necessary, but not sufficient for receptor cleavage, which requires the entire extracellular stalk. Thus, an alternatively spliced EMR2 isoform with a truncated stalk fails to undergo proteolytic cleavage. Alternative splicing therefore provides a means to regulate GPS cleavage, producing receptors with two distinct structures. [Copyright &y& Elsevier]

MacB is an ABC-type membrane protein that exports only macrolide compounds containing 14- and 15-membered lactones, cooperating with a membrane fusion protein, MacA, and a multifunctional outer membrane channel, TolC. We determined the membrane topology of MacB by means of site-specific competitive chemical modification of single cysteine mutants. As a result, it was revealed that MacB is composed of four transmembrane (TM) segments with a cytoplasmic N-terminal nucleotide binding domain of about 270 amino acid residues and a periplasmic large hydrophilic polypeptide between TM segments 1 and 2 of about 200 amino acid residues. [Copyright &y& Elsevier]

Transhydrogenase, in animal mitochondria and bacteria, couples hydride transfer between NADH and NADP+ to proton translocation across a membrane. Within the protein, the redox reaction occurs at some distance from the proton translocation pathway and coupling is achieved through conformational changes. In an ‘open’ conformation of transhydrogenase, in which substrate nucleotides bind and product nucleotides dissociate, the dihydronicotinamide and nicotinamide rings are held apart to block hydride transfer; in an ‘occluded’ conformation, they are moved into apposition to permit the redox chemistry. In the two monomers of transhydrogenase, there is a reciprocating, out-of-phase alternation of these conformations during turnover. [Copyright &y& Elsevier]

Low-resolution experiments suggest that most membrane helices span over 17-25 residues and that most loops between two helices are longer than 15 residues. Both constraints have been used explicitly in the development of prediction methods. Here, we compared the largest possible sequence-unique data sets from high- and low-resolution experiments. For the high-resolution data, we found that only half of the helices fall into the expected length interval and that half of the loops were shorter than 10 residues. We compared the accuracy of detecting short loops and long helices for 28 advanced and simple prediction methods: All methods predicted short loops less accurately than longer ones. In particular, loops shorter than 7 residues appeared to be very difficult to detect by current methods. Similarly, all methods tended to be more accurate for longer than for shorter helices. However, helices with more than 32 residues were predicted less accurately than all other helices. Our findings may suggest particular strategies for improving predictions of membrane helices. [ABSTRACT FROM AUTHOR]

Methods that predict membrane helices have become increasingly useful in the context of analyzing entire proteomes, as well as in everyday sequence analysis. Here, we analyzed 27 advanced and simple methods in detail. To resolve contradictions in previous works and to reevaluate transmembrane helix prediction algorithms, we introduced an analysis that distinguished between performance on redundancy-reduced high- and low-resolution data sets, established thresholds for significant differences in performance, and implemented both per-segment and per-residue analysis of membrane helix predictions. Although some of the advanced methods performed better than others, we showed in a thorough bootstrapping experiment based on various measures of accuracy that no method performed consistently best. In contrast, most simple hydrophobicity scale-based methods were significantly less accurate than any advanced method as they overpredicted membrane helices and confused membrane helices with hydrophobic regions outside of membranes. In contrast, the advanced methods usually distinguished correctly between membrane-helical and other proteins. Nonetheless, few methods reliably distinguished between signal peptides and membrane helices. We could not verify a significant difference in performance between eukaryotic and prokaryotic proteins. Surprisingly, we found that proteins with more than five helices were predicted at a significantly lower accuracy than proteins with five or fewer. The important implication is that structurally unsolved multispanning membrane proteins, which are often important drug targets, will remain problematic for transmembrane helix prediction algorithms. Overall, by establishing a standardized methodology for transmembrane helix prediction evaluation, we have resolved differences among previous works and presented novel trends that may impact the analysis of entire proteomes. [ABSTRACT FROM AUTHOR]

Transmembrane potentials play important but poorly understood roles in many biological processes, including signal sequence-mediated protein translocation across bacterial membranes. In this study we applied Monte Carlo techniques to simulate the way the potential acts on a signal sequence. The simulations demonstrate that in the absence of a potential the signal sequence prefers insertion in both helical hairpin and transmembrane α-helical conformations. However, in the presence of a potential only the transmembrane α-helical conformation is the state of lowest energy for the signal sequence. From these results it is concluded that the membrane potential stabilizes the transmembrane orientation of a signal sequence, explaining the membrane potential dependence of preprotein translocation. [Copyright &y& Elsevier]

The neuropeptide Y (NPY) receptor Y2 antagonist BIIE0246 has sub-nanomolar affinity for the human Y2 (hY2) receptor but binds very poorly to chicken Y2 (chY2) with micromolar affinity. Sequence comparisons identified several amino acids for investigation by mutagenesis. Reciprocal mutagenesis between hY2 and chY2 revealed that three of these, individually and in combination, are important for BIIE0246 binding, namely positions Gln135 in transmembrane (TM) 3, Leu227 in TM5, and Leu284 in TM6. Mutagenesis of hY2 to the corresponding amino in chY2 (generating hY2[Q135H,L227Q,L284F]) made the affinity of BIIE0246 as low as for chY2. Introduction into chY2 of the three human residues resulted in antagonist affinity almost as high as for hY2. To distinguish between direct and indirect effects, each of the three residues in hY2 was replaced with alanine. BIIE0246 bound with 28-fold lower affinity to hY2[L227A], suggesting the Leu227 interacts directly with the antagonist. The other two alanine mutants bound with unaltered affinity, suggesting that the corresponding chY2 residues abolish binding through steric hindrance or charge repulsion. Thus, three amino acid residues can in an additive manner completely account for the difference in antagonist binding between the hY2 and chY2 receptors. These results will be useful for construction of three-dimensional models of the widely divergent NPY receptor subtypes. [Copyright &y& Elsevier]